The Black Widow spider is one of the few spider species that are poisonous to humans. The bite of this spider seriously endangers human health and results in a systemic poisoning syndrome called latrodectism. For the very young, the elderly and those with cardiovascular disease, the envenomation can be fatal. Black Widow spider venom contains alpha-latrotoxin, a neurotoxin active in vertebrates. It is a specific presynaptic toxin that acts by stimulating neurotransmitter release. Prolonged exposure to the toxin also results in nerve terminal degeneration. This proposal aims to clarify the molecular mechanisms leading to synaptic impairment by alpha-latrotoxin. This information may help to elucidate the synaptic basis of other neurotoxic substances, to suggest appropriate therapies, as well as to add to understanding of the roles for nerve terminal proteins in normal synaptic function. The proposed experiments will study in detail the structure and function of the protein components of the brain high affinity alpha-latrotoxin receptor which has been purified in previous studies. The specific aims of the proposed experiments are: 1. To characterize the polypeptide composition of the high affinity alpha-latrotoxin receptor and to identify the alpha-latrotoxin-binding protein(s). The purified receptor preparations contain a large molecular weight protein (neurexin) present in two isoforms of Mr 200,000 and 160,000 bound to a small polypeptide of Mr 29,000 (neurexophilin). Molecular cloning of neurexin led to the discovery of a novel family of synapse-specific membrane proteins that exist in multiple isoforms. The alpha-latrotoxin receptor was preliminarily identified as neurexin Ia. The proposed experiments are to further study the composition of the receptor and to test whether other neurexins also bind alpha-latrotoxin. 2. To analyze the structure of neurexophilin and to study the structural requirements of the formation of neurexin-neurexophilin complexes. Neurexophilin was cloned and the preliminary sequencing data suggest that this protein is a novel secreted neuropeptide. The binding of recombinant deletion mutants of neurexophilin to neurexin Ia and its proteolytic fragments will be analyzed to identify the binding domains. Neurexophilin will be further characterized biochemically and its affinity to other neurexins will be estimated. 3. To compare the cellular and subcellular distribution of alpha-latrotoxin binding sites, neurexins and neurexophilin in primary neuronal cell cultures and in the brain. These experiments will hep us to understand whether neurexophilin is a subunit or an endogenous ligand of the alpha-latrotoxin receptor. 4. To analyze the effects of neurexophilin on physiological and alpha-latrotoxin-evoked neurotransmitter release. Together, these experiments will provide a molecular description of the pathways of the Black Widow spider venom toxicity.